molecular epidemiology and pathology of spirorchiid infection in green sea turtles chelonia mydas

Accepted Manuscript Molecular epidemiology and pathology of spirorchiid infection in green sea turtles (Chelonia mydas) Phoebe A Chapman, Helen Owen, Mark Flint, Ricardo J Soares Magalhães, Rebecca J Traub, Thomas H Cribb, Myat T Kyaw-Tanner, Paul C Mills PII: S2213-2244(16)30046-3 DOI: 10.1016/j.ijppaw.2017.03.001 Reference: IJPPAW 186 To appear in: International Journal for Parasitology: Parasites and Wildlife Received Date: 21 October 2016 Revised Date: 28 February 2017 Accepted Date: March 2017 Please cite this article as: Chapman, P.A., Owen, H., Flint, M., Soares Magalhães, R.J., Traub, R.J., Cribb, T.H., Kyaw-Tanner, M.T., Mills, P.C., Molecular epidemiology and pathology of spirorchiid infection in green sea turtles (Chelonia mydas), International Journal for Parasitology: Parasites and Wildlife (2017), doi: 10.1016/j.ijppaw.2017.03.001 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Molecular epidemiology and pathology of spirorchiid infection in green sea turtles (Chelonia mydas) Phoebe A Chapmana, Helen Owena, Mark Flinta,b, Ricardo J Soares Magalhãesc,d, Rebecca J Traube, Thomas H Cribbf, Myat T Kyaw-Tannera, Paul C Millsa a University of Queensland, Gatton, Queensland, AUSTRALIA; b for Conservation, Apollo Beach, Florida, USA; c Veterinary-Marine Animal Research, Teaching and Investigation, School of Veterinary Science, The SC School of Forest Resources and Conservation, University of Florida, The Florida Aquarium’s Center M AN U UQ Spatial Epidemiology Laboratory, School of Veterinary Science, University of Queensland, 10 Gatton, Queensland, AUSTRALIA; 11 d 12 Queensland, South Brisbane, Queensland, Australia 13 e 14 AUSTRALIA; 15 f Children’s Health and Environment Program, Child Health Research Centre, The University of TE D Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, Victoria, School of Biological Science, The University of Queensland, St Lucia, Queensland, AUSTRALIA EP 16 RI PT Corresponding author: P A Chapman, School of Veterinary Science, University of Queensland 18 Gatton Campus, Gatton, Queensland, AUSTRALIA 4343 Email: 19 phoebe.chapman@uqconnect.edu.au AC C 17 ACCEPTED MANUSCRIPT ABSTRACT 21 Spirorchiid blood fluke infections affect endangered turtle populations globally, and are reported as a 22 common cause of mortality in Queensland green sea turtles Both the flukes and their ova are 23 pathogenic and can contribute to the stranding or death of their host Of particular interest are ova- 24 associated brain lesions, which have been associated with host neurological deficits Accurate 25 estimations of disease frequency and the relative effect of infection relating to different spirorchiid 26 species are made difficult by challenges in morphological identification of adults of some genera, and 27 a lack of species-level identifying features for ova A new specifically designed molecular assay was 28 used to detect and identify cryptic spirorchiids and their ova in Queensland green sea turtle tissues 29 collected from 2011 to 2014 in order to investigate epidemiology, tissue tropisms and pathology Eight 30 spirorchiid genotypes were detected in 14 distinct tissues, including multiple tissues for each We 31 found no evidence of a characteristic pathway of the eggs to the exterior; instead the results suggest 32 that a high proportion of eggs become lost in dead-end tissues The most common lesions observed 33 were granulomas affecting most organs with varying severity, followed by arteritis and thrombi in the 34 great vessels The number of spirorchiid types detected increased with the presence and severity of 35 granulomatous lesions However, compared with other organs the brain showed relatively low levels 36 of spirorchiid diversity An inverse relationship between host age and spirorchiid diversity was evident 37 for the liver and kidneys, but no such relationship was evident for other organs Molecular data in this 38 study, the first of its kind, provides the first species-level examination of spirorchiid ova and 39 associated pathology, and paves the way for the future development of targeted ante-mortem 40 diagnosis of spirorchiidiasis 41 KEYWORDS 42 Spirorchiidiasis, Chelonia mydas, epidemiology, pathology, tissue tropisms, terminal restriction 43 fragment length polymorphism AC C EP TE D M AN U SC RI PT 20 ACCEPTED MANUSCRIPT 44 INTRODUCTION Each year, hundreds of marine turtles are reported stranded or dead on the east coast of 46 Queensland, Australia (Flint et al 2015a; Queensland Department of Environment and Heritage 47 Protection 2015) From 2009 to 2014, an annual average of 1,152 strandings or mortalities were 48 recorded; the majority of these were green sea turtles (Flint et al 2015a), which are currently listed as 49 Endangered by the International Union for the Conservation of Nature Disease is among the most 50 commonly recorded causes of stranding or mortality (Flint et al 2009a; Meager and Limpus 2012), 51 but infectious causes of turtle mortality are poorly understood Parasites, particularly spirorchiid blood 52 flukes and the coccidian Caryospora cheloniae, are noted for their capacity to cause disease (Gordon 53 et al 1998; Santoro et al 2007; Flint et al 2010; Stacy et al 2010) and mortality (Gordon et al 1993; 54 Flint et al 2010; Chapman et al 2016a) Of the two, spirorchiids are the more common and 55 widespread 56 Spirorchiid flukes infect all major organs, and both adults and ova can have deleterious effects that 57 may contribute to the stranding or death of their host (Glazebrook et al 1989; Glazebrook and 58 Campbell 1990; Aguirre et al 1998; Gordon et al 1998; Work et al 2004; Work et al 2005; Santoro 59 et al 2007; Flint et al 2010; Stacy et al 2010; Flint et al 2015b) In Queensland, spirorchiidiasis is 60 considered the most significant infectious disease among sea turtles (Flint et al 2010) The earliest 61 studies in the region found that spirorchiids were present in 40.9% to 72.2% of wild sea turtles 62 (Glazebrook et al 1989; Glazebrook and Campbell 1990) and were associated with a range of lesions 63 and general debilitation In 1998, Gordon et al (1998) found spirorchiids to be the primary cause of 64 death in 10% of locally stranded green sea turtles and a severe problem in a further 30%, with an 65 overall infection rate of 98% on the basis of histopathology Spirorchiids were also found to be the 66 most common cause of death (41.8%) in Queensland green sea turtles between 2006 and 2009 (Flint 67 et al 2010) Spirorchiids maintain a high prevalence in turtles globally, including Hawaii (Graczyk et 68 al 1995; Aguirre et al 1998; Work et al 2005; Work et al 2015), the eastern United States (Stacy et 69 al 2010) and South America (Santoro et al 2007) Incidental, asymptomatic infections are common 70 (Gordon et al 1998; Santoro et al 2007; Flint et al 2010; Stacy et al 2010; Work et al 2015) 71 However, the occurrence of severe disease appears to vary spatially; while spirorchiidiasis is often a 72 primary cause of death in Australian sea turtles (Flint et al 2010), it is less frequently fatal in turtles AC C EP TE D M AN U SC RI PT 45 ACCEPTED MANUSCRIPT from the south-eastern United States (Stacy et al 2010) and Hawaii (Work et al 2015) The reasons 74 behind these geographic variations are unexplained 75 Accurate estimations of prevalence and the relative impact of individual spirorchiid species are 76 constrained by two factors First, the adults of some species are microscopic and show an apparent 77 predilection for small blood vessels, making them very difficult to detect and collect intact (Gordon et 78 al 1998; Stacy et al 2010) Secondly, ova can only be categorised into one of three broad 79 morphological types, and identification to species level is not possible Given the limitations of 80 traditional gross and microscopy based methods in this field, molecular techniques may contribute 81 substantially to our knowledge of the parasite and the disease Molecular tools have superior 82 sensitivity and specificity relative to traditional microscopic/histologic identification (McManus and 83 Bowles 1996; Ndao 2009; Chapman et al 2016b), and the additional advantage of having potential 84 for diagnostic applications in live turtles Such approaches can therefore increase capacity to detect 85 and identify parasites and explore their connection with pathology Reports of pathology associated 86 with ova are so far restricted to generalised comments on associated pathology and rarely attempt to 87 associate lesions with particular species Given that spirorchiid ova are almost universally present and 88 often present in the apparent absence of adult flukes (Flint et al 2010; Stacy et al 2010), they require 89 detailed investigation of their impacts and association with pathology Recently, a new molecular 90 assay was developed and validated for the identification of spirorchiids and their ova in green sea 91 turtle tissues (Chapman et al 2016b) enabling the collection of data of a kind that has, to date, been 92 unavailable 93 This paper aims to improve understanding of spirorchiidiasis in turtle populations of Australia and 94 other regions around the globe by examining and quantifying infection rates, tissue tropisms, and 95 predisposing host factors using the newly developed test (Chapman et al 2016b) AC C EP TE D M AN U SC RI PT 73 96 97 MATERIALS AND METHODS 98 2.1 99 Green sea turtle carcasses were obtained from the wildlife rehabilitation facilities Sea Life Underwater 100 Study population World (Mooloolaba) and Australia Zoo Wildlife Hospital (Beerwah) as well as government agencies ACCEPTED MANUSCRIPT (Queensland Parks and Wildlife Service – QPWS) between 2011 and 2015 Prior to necropsy turtles 102 were stored in refrigeration for a maximum of days, or otherwise frozen Turtles were mainly from 103 two broad locations: the central Queensland region from Gladstone Port (23.8251˚S, 151.2975˚E), 104 nearby islands (Quoin, Facing and Boyne) south to the town of 1770 (24.1594˚S, 151.8658˚E), and 105 the southern Queensland area between Hervey Bay (25.2538˚S, 152.8605˚E) and Ormiston 106 (27.5112˚S, 153.2675˚E) One further turtle was collected from the Townsville area (19.2372˚S, 107 146.8985˚E) in northern Queensland These areas encompass three of several ‘hotspots’ for marine 108 turtle strandings on Australia’s east coast (Queensland Department of Environment and Heritage 109 Protection 2013) and therefore provided an opportunity to investigate the role of diseases in 110 strandings Turtles were classified into age groups using the size criteria utilised by Flint et al (2010) 111 and body condition scores were estimated visually and assigned using the criteria described by Flint 112 et al (2009b) to provide an overall post mortem health profile 113 2.2 114 Turtles were necropsied between 2011 and early 2015 using standard methods for sea turtle post 115 mortem examination (Flint et al 2009b) During necropsy, adult flukes were collected and identified 116 using molecular and morphological methods as described by Chapman et al (2015) Samples of 117 turtle tissues were collected for molecular analysis For tubiform organs such as the gastrointestinal 118 tract and major blood vessels, a cross section was taken to include all cell layers When sampling the 119 gastrointestinal tract, samples were collected from the stomach or small intestine, depending on 120 evidence of spirorchiid infection i.e visible egg deposition Samples were stored in 70% ethanol at 121 4°C before analysis using PCR and T-RFLP as described by Chapman et al (2016b) This method 122 involved an initial multiplex PCR to detect and identify spirorchiid genera based on the 28S gene, 123 followed by a second round of singleplex reactions using fluorescently tagged genus specific primers 124 to produce PCR products for species level analysis through restriction nuclease digestion and 125 capillary electrophoresis A total of twelve genotypes were tested for Many of these genotypes 126 corresponded with known species, though several were not able to be matched to an existing 127 morphological or molecular identification; regardless, they were interpreted as distinct species based 128 on the level of genetic variation observed between types (Chapman et al 2016b) Negative results 129 from first round multiplex PCR were validated by assessing the presence of amplifiable DNA with M AN U AC C EP TE D Parasitological methods SC RI PT 101 ACCEPTED MANUSCRIPT universal eukaryote 18S primers (Fajardo et al 2008; Chapman et al 2016b) For unfrozen 131 carcasses with minimal decomposition, further samples were collected and fixed in 10% neutral 132 buffered formalin for histological examination to correlate turtle health with parasitic effect These 133 samples were embedded in paraffin wax prior to being sectioned at µm and stained with 134 haematoxylin and eosin (HE) Sections were examined by a specialist veterinary pathologist 135 2.3 136 The presence of spirorchiid ova in tissues was assessed by histology, along with the presence of 137 associated inflammatory lesions (e.g granulomas) Granulomas were graded based on relative 138 severity, accounting for size and number of lesions as well as disruption to surrounding cellular 139 architecture Grading was based on the methodology described by Flint et al (2010), however, a five 140 point scale was used with a score of designated for mild, for moderate and for severe lesions; 141 scores of and were used in cases where lesions did not clearly meet the criteria either side, or 142 varied in severity across the section 143 2.4 144 The proportion of organs infected with each spirorchiid genus was estimated from PCR data, and 145 genotypes from T-RFLP results Samples with PCR results but without species level T-RFLP were 146 omitted from species level calculations For the purpose of this study, Hapalotrema, Learedius and 147 Amphiorchis were grouped together due to their genetic and morphological similarity and comparable 148 reported site tropisms, with genus level proportions calculated as one 149 We compared the occurrence of granulomas within each organ type by age, sex, body condition and 150 infection type (single or multi species infections) using the Fisher’s exact test (95% confidence 151 interval) 152 We investigated the association between granuloma presence in brain samples (outcome) and 153 spirorchiid infection type (exposure of interest) using multivariable generalised linear model (GLM) 154 The model was adjusted for the effect of age, sex (female – 0, male – 1), body condition and Bernoulli 155 distributed residuals (binomial family) with a logit link function Each sample was categorised as either 156 single infection (i.e one spirorchiid genotype present - 0) or multiple infection (i.e two or more 157 genotypes - 1) Mature and large immature classes were combined for analysis, resulting in two age RI PT 130 AC C EP TE D Statistical analyses M AN U SC Histopathological methods ACCEPTED MANUSCRIPT groups of 65 cm (mature and large immature - 1) 159 Turtles judged to be in poor or very poor body condition were combined into one category (0) 160 representing turtles with deemed chronic debility, while those assessed as good or fair formed the 161 second category (1) Analyses of spirorchiid occurrence were undertaken at both the genus and 162 species level The effect size of each of the predictor variables were expressed as odds ratios with 163 95% confidence intervals The Mann-Whitney U- test was used to compare the average number of 164 spirorchiids in relation to age, sex and body condition in other organs All analyses were performed 165 using STATA version 13.1 (StataCorp, Texas, USA) 166 2.5 167 All work described above was completed under approval SVS/037/11/ARC/DERM/AUSTZOO issued 168 by the University of Queensland Animal Ethics Committee Approval to undertake marine research 169 activities was granted through Queensland State Government Marine Parks permit QS2011/CVL1414 170 and Scientific Purposes permit WISP09021911 M AN U SC Ethical Standards RI PT 158 171 172 TE D RESULTS 173 3.1 174 Necropsies were completed on a total of 51 turtles Various tissue samples from 39 turtles were 175 examined histologically, while tissue samples from 44 animals were collected for molecular 176 characterisation of spirorchiids A summary of host characteristics and tissue samples collected is 177 provided in Table 178 3.2 EP AC C 179 Dataset for analysis Parasitological findings 3.2.1 Adult flukes 180 Adult flukes were found in 16/51 turtles (Table 2) The majority of flukes found were larger worms, i.e 181 species of Hapalotrema and Learedius The most common was H pambanensis, which was found in 182 the heart of six turtles, with additional flukes found in the aortic vessels (left or pulmonary) in two of 183 these cases In two additional turtles, they were recovered from the body cavity, presumably after ACCEPTED MANUSCRIPT having been released from the circulatory system during necropsy Hapalotrema postorchis showed a 185 particular affinity for the major vessels (five turtles) while L learedi was found in the heart of two 186 turtles, with further specimens also present in the lung of one In most cases multiple flukes were 187 collected, with over 80 recovered in severe infections (Table 2) 188 Adult flukes confirmed as Carettacola sp were found in six turtles, and were recovered from sites 189 including the liver (three turtles), pancreas (one turtle), and body cavity (four turtles) In some 190 instances, these flukes were unable to be identified to species level on account of the samples being 191 broken or in poor condition However, flukes from three turtles were identified as C hawaiiensis 192 based on either morphological or molecular characteristics Others were genetically distinct but did 193 not match limited sequences available in Genbank One fluke retrieved from the body cavity was 194 initially identified as a Carettacola sp but sequence data indicates that it was likely to relate to an 195 unknown spirorchiid genus (Genbank KU600078.1) 196 Difficulties were also experienced in identifying Neospirorchis spp that were retrieved from the heart, 197 brain, lung and body cavity of five turtles, due to damaged and broken samples and a lack of genetic 198 data available in public databases Flukes from Genotype were collected from the brain and body 199 cavity, Genotype from the heart, and Genotype from the lungs SC M AN U 3.2.2 TE D 200 RI PT 184 Molecular detection - tissue samples Spirorchiid infections were detected in 142/148 tissue samples, encompassing 43/44 turtles from 202 which samples were tested Of six that failed to give a positive PCR result, four were validated as 203 genuine negative results, while the remaining two were discarded owing to failure of the generic 204 eukaryote 18S PCR assay to amplify genomic host DNA Full molecular characterisation was 205 achieved for 129 samples; the remainder were limited to initial multiplex PCR characterisation or, in 206 some cases, multiplex PCR plus T-RFLP results for one primer set only, most likely due to DNA 207 degradation 208 A total of eight distinct spirorchiid genotypes (interpreted as representing distinct species) were 209 detected The occurrence of each spirorchiid genotype as detected by PCR and T-RFLP is 210 summarised in Table Overall, Neospirorchis was the most common genus Of samples that were 211 successfully characterised to species level, between 80% and 100% of each organ (excepting the gall AC C EP 201 ACCEPTED MANUSCRIPT The T-RFLP assay detected Neospirorchis spp in the great majority of samples Tissues from all but 324 one of the 44 turtles tested positive, with Neospirorchis Genotype being most common Meanwhile, 325 Carettacola spp were relatively rare The ubiquitous nature of Neospirorchis infections in green sea 326 turtles has previously been noted in Queensland (Gordon et al 1998) and Florida (Stacy et al 2010) 327 Meanwhile, Work et al (2005) noted the low prevalence of Carettacola infections in Hawaiian green 328 turtles, and although Stacy et al (2010) found C bipora in loggerhead turtles (Caretta caretta) from 329 Florida, they did not find any Carettacola species in green turtles Life cycle factors may contribute to 330 the difference in frequency of Neospirorchis and Carettacola infections, e.g relative fecundity and/or 331 abundance of intermediate hosts We also considered whether the lower detection rate for 332 Carettacola found here might relate to the sensitivity of the primers targeting this genus However, 333 although the validation of this assay (Chapman et al 2016b) found that the Carettacola primer pair 334 had a higher detection limit than those for other genera, it was still sufficient to detect low numbers of 335 eggs Conversely, adult Carettacola were recovered more often than adult Neospirorchis, likely a 336 result of differences in size, body morphology, and microhabitat usage 337 The number of spirorchiid species detected in the brain was found to be significantly greater when 338 granulomas were present Also, when all granulomatous tissues were considered, the average 339 number of species detected increased with the severity of lesions One possible mechanism 340 contributing to this finding is the onset of local alteration of blood flow that occurs in response to the 341 initial embolization of ova, which in turn predisposes the accumulation of ova of further species within 342 the lesion General debility of the host permitting infection by a larger number of species is also likely 343 to contribute to the number of species observed in a lesion, and may simultaneously facilitate 344 progression to significant inflammatory responses No individual species appeared to be 345 disproportionately associated with granulomatous lesions Neospirorchis Genotype was detected in 346 all brains with granulomas, sometimes as the only species present, but was also found in brains 347 showing no observable granuloma formation It is therefore not possible to implicate this genotype 348 alone as a cause of lesions The development of inflammatory lesions is likely to be influenced by 349 multiple factors (e.g individual response to insult, length of period of infection, environmental 350 stressors, injury or coinfection with other pathogens) The relationship between brain lesions and 351 clinical disease is a matter of interest because clinical neurological cases in recent times in Australia 352 and the United States have been presumptively diagnosed as spirorchiidiasis Neurological infection AC C EP TE D M AN U SC RI PT 323 13 ACCEPTED MANUSCRIPT with Neospirorchis spp was associated with a mass mortality event in loggerhead turtles (Caretta 354 caretta) in Florida in the early 2000s (Jacobson et al 2006) however the exact nature of the parasite’s 355 role was not resolved Neurological impairment has also been observed alongside spirorchiid- 356 associated brain lesions in green sea turtles from Queensland (Flint et al 2010) and Western 357 Australia (Raidal et al 1998) Such neurological deficits, while injurious in themselves, may also 358 predispose the animal to misfortunes such as boat strike or predation The availability of molecular 359 tools enables greater capacity to determine which Neospirorchis species are associated with 360 neurological lesions The methodology used here identifies the species involved, paving the way for 361 the development of specific anti-mortem diagnostics In this study, the species associated with the 362 most severe neurological lesions observed are commonly present in turtles of all age and health 363 statuses Continued surveillance and development of quantitative ante-mortem testing for implicated 364 species is likely to assist in unravelling the factors that lead to severe clinical effects 365 In this study, overall spirorchiid diversity and the prevalence of individual species (particularly of 366 species within the Hapalotrema/Learedius group) generally decreased with age An exception was H 367 mistroides, which, in contrast to other Hapalotrema species, was detected more often in mature turtles 368 than small immature ones, potentially due to greater accumulated exposure in large immature or 369 mature turtles The overall tendency for parasitic infections to decrease with age is likely to reflect 370 progressive development of immunity after immunologically naïve small immature turtles are exposed 371 to spirorchiids at the time of recruitment to neritic habitats (Work et al 2005; Flint et al 2010) 372 Acquired immunity to blood flukes in various vertebrate hosts has been routinely observed For 373 example, infections of the related Schistosoma spp in species such as humans (Cheever et al 1977; 374 Agnew et al 1996; Drake and Bundy 2001), primates (Cheever et al 1974; He et al 1992; Li et al 375 2015), water buffalo (Li 2014), cattle and pigs (Vercruysse and Gabriel 2005) generally decrease in 376 intensity as the host ages Meanwhile, the ability of poikilotherms to develop immune responses to 377 blood flukes is demonstrated by the progressive development of humoral responses by Pacific Bluefin 378 tuna (Thunnus maccoyii) to aporocotylid flukes (Cardicola spp.) (Aiken et al 2008; Kirchhoff et al 379 2012; Polinski et al 2014) Despite this, green turtle populations in the Pacific (Work et al 2005; Flint 380 et al 2010) and the Atlantic (Santoro et al 2007; Stacy et al 2010) show different relationships 381 between age and apparent susceptibility to spirorchiid infection The factors causing variations in 382 epizootiology between populations in different geographic regions remain unclear Potential factors AC C EP TE D M AN U SC RI PT 353 14 ACCEPTED MANUSCRIPT include different spirorchiid species assemblages, and environmental factors which may cause 384 differential exposure to infective stages (e.g microhabitat variations and presence of intermediate 385 hosts), or cause stress to hosts (e.g pollutants and poor water quality), resulting in increased 386 susceptibility to disease (Flint et al 2015b) Thus, spirorchiidiasis requires analysis in the context of 387 distinct host species, parasite species and habitats 388 Possible future research directions following up from our findings could include quantitative 389 assessment of the number of spirorchiid eggs of each species or genotype in a sample This 390 approach would allow estimation of the relative contribution of each type to lesion development 391 Currently, quantitative molecular methods are largely restricted to quantitative real-time PCR (qPCR) 392 However, the development of species/genotype specific primer/probe sets to target each spirorchiid 393 would prove difficult given relatively low observed levels of interspecific variation in commonly 394 examined spirorchiid genes and the high number of target genotypes Visual methods of quantitation 395 i.e histology or tissue digestion and microscopic counts can produce data only to a genus level In the 396 case of histology, this can be difficult to achieve accurately due to sectional angles and degeneration 397 of eggs 398 As discussed, low numbers of samples were available overall and disproportionate numbers of 399 samples came from small immature turtles The relationship between age, the number of spirorchiid 400 species present and presence of lesions varied between organs, which is likely due to low sample 401 numbers as evidenced by broad confidence intervals However, genus level infection rates and tissue 402 tropisms were generally reflective of those reported previously Full comparable sets of tissue 403 samples were not available from all turtles, with infection rates calculated per organ rather than at the 404 host level, but this in itself provides insight into tissue tropisms Some parasite species detected here 405 are yet to be fully identified due to a lack of suitable samples for morphological characterisation, either 406 due to their cryptic and fragile nature or freezing of the turtle prior to collection for necropsy No effect 407 on molecular results due to freezing is likely, and it is anticipated that the parasites will be definitively 408 identified and/or described in future studies Until recently, a lack of life cycle data has constrained our 409 understanding of disease transmission and ability to control infections, however, recent discoveries in 410 this area (Cribb et al 2017), paired with specific molecular diagnostic tools, will lead to improved 411 knowledge of disease epidemiology AC C EP TE D M AN U SC RI PT 383 15 ACCEPTED MANUSCRIPT This study has provided the first molecular study of the distribution of spirorchiids in the tissues of 413 green sea turtles, and provides data at a level of specificity not previously attainable It demonstrates 414 that young turtles in this region are likely to be infected with the greatest diversity of spirorchiids, and 415 that at least in the brain, there is a relationship between the presence and severity of granulomatous 416 lesions and the number of spirorchiid species present It is clear that variation in disease presentation 417 between geographic regions is significant, yet not understood The influence of spatially fluctuating 418 environmental factors and stressors is of particular interest in understanding the triggers for the 419 development of clinical disease, as is the elucidation of further spirorchiid life cycles and the 420 development of ante-mortem diagnostics for investigation and monitoring of live turtle populations SC RI PT 412 421 ACKNOWLEDGEMENTS 423 This work was supported by the Australian Research Council (Linkage Grant number LP110100569) 424 We acknowledge staff at the wildlife rehabilitation facilities Australia Zoo (Beerwah, QLD) and 425 Underwater World (Mooloolaba, QLD) as well as the Queensland Department of Environment and 426 Heritage Protection (EHP) and Queensland Parks and Wildlife Service (QPWS) for facilitating access 427 to dead turtles for examination Chris Cazier (histology) and the Animal Genetics Laboratory (capillary 428 electrophoresis) at The University of Queensland’s School of Veterinary Science provided technical 429 services Thanks to Dr Paul Eden for initial work in sample collection 430 EP TE D M AN U 422 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infected with Chinese mainland strain of Schistosoma japonicum Southeast Asian J Trop Med Public Health 23, pp 254-260 AC C 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 17 ACCEPTED MANUSCRIPT EP TE D M AN U SC RI PT Jacobson E.R., Homer B.L., Stacy B.A., Greiner E.C., Szabo N.J., Chrisman C.L., Origgi F., Coberley S., Foley A.M., Landsberg J.H., Flewelling L., Ewing R.Y., Moretti R., Schaf S., Rose C., Mader D.R., Harman G.R., Manire C.A., Mettee N.S., Mizisin A.P., Shelton G.D., 2006 Neurological disease in wild loggerhead sea turtles Caretta caretta Dis Aquat Org 70, pp 139-154 Kirchhoff N.T., Leef M.J., Valdenegro V., Hayward C.J., Nowak B.F., 2012 Correlation of humoral immune response in southern bluefin tuna, T maccoyii, with infection stage of the blood fluke, Cardicola forsteri PLoS ONE 7, pp e45742 Li X.-H., Xu Y.-X., Vance G., Wang Y., Lv L.-B., van Dam G.J., Cao J.-P., Wilson R.A., 2015 Evidence that rhesus macaques self-cure from a Schistosoma japonicum infection by disrupting worm esophageal function: a new route to an effective vaccine? PLoS Negl Trop Dis 9, pp e0003925 Li Y.-S., 2014 The Schistosoma japonicum self-cure phenomenon in water buffaloes: potential impact on the control and elimination of schistosomiasis in China Int J Parasitol 44, pp 167171 McManus D.P., Bowles J., 1996 Molecular genetic approaches to parasite identification: their value in diagnostic parasitology and systematics Int J Parasitol 26, pp 687-704 Meager J.J., Limpus C.J., 2012 Marine wildlife stranding and mortality database annual report 2011 III Marine Turtle vol Department of Environment and Heritage Protection,, Queensland Ndao M., 2009 Diagnosis of parasitic diseases: old and new approaches Interdiscip Perspect Infect Dis 2009, pp Polinski M., Shirakashi S., Bridle A., Nowak B., 2014 Transcriptional immune response of cagecultured Pacific bluefin tuna during infection by two Cardicola blood fluke species Fish Shellfish Immunol 36, pp 61-67 Queensland Department of Environment and Heritage Protection 2013 Marine wildlife strandings data http://www.ehp.qld.gov.au/wildlife/caring-for-wildlife/marine-strandings-data.html Accessed 24 August 2016 Queensland Department of Environment and Heritage Protection 2015 Marine strandings update 2015 https://www.ehp.qld.gov.au/wildlife/caring-for-wildlife/marine-strandingsupdate.html Accessed May 2016 Raidal S.R., Ohara M., Hobbs R.P., Prince R.I.T., 1998 Gram-negative bacterial infections and cardiovascular parasitism in green sea turtles (Chelonia mydas) Aust Vet J 76, pp 415-417 Santoro M., Morales J.A., Rodriguez-Ortiz B., 2007 Spirorchiidiosis (Digenea: Spirorchiidae) and lesions associated with parasites in Caribbean green turtles (Chelonia mydas) Vet Rec 161, pp 482-486 Stacy B.A., Foley A.M., Greiner E., Herbst L.H., Bolten A., Klein P., Manire C.A., Jacobson E.R., 2010 Spirorchiidiasis in stranded loggerhead Caretta caretta and green turtles Chelonia mydas in Florida (USA): host pathology and significance Dis Aquat Org 89, pp 237-259 Vercruysse J., Gabriel S., 2005 Immunity to schistosomiasis in animals: an update Parasite Immunol 27, pp 289-295 Work T.M., Balazs G.H., Rameyer R.A., Morris R.A., 2004 Retrospective pathology survey of green turtles Chelonia mydas with fibropapillomatosis in the Hawaiian Islands, 1993-2003 Dis Aquat Org 62, pp 163-176 Work T.M., Balazs G.H., Schumacher J.L., Marie A., 2005 Epizootiology of spirorchiid infection in green turtles (Chelonia mydas) in Hawaii J Parasitol 91, pp 871-876 Work T.M., Balazs G.H., Summers T.M., Hapdei J.R., Tagarino A.P., 2015 Causes of mortality in green turtles from Hawaii and the insular Pacific exclusive of fibropapillomatosis Dis Aquat Org 115, pp 103-110 AC C 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 18 ACCEPTED MANUSCRIPT Legends to Figures 545 Fig Lesions associated with spirorchiid blood flukes in Chelonia mydas a) Mild (score = 546 1) granulomatous lesions (G) and lymphocytic inflammation within the cerebral meninges of 547 an adult green turtle, centred on a brown-shelled fluke ovum HE stain, scale bar = 125 µm 548 b) Moderate (score = 3) granulomatous lesions (G) and lymphocytic inflammation within the 549 cerebral meninges of an adult green turtle, centred on several brown-shelled fluke ova HE 550 stain, scale bar = 350 µm c) Severe (score = 5) granulomatous lesions (G) and lymphocytic 551 inflammation within the meninges of a small immature green turtle, centred on multiple 552 numerous fluke ova HE stain, scale bar = 350 µm d) Severe (score = 5) granulomatous 553 lesion (G) with necrotic centre protruding into the lumen of the aorta of an adult green turtle, 554 with dense lymphocytic inflammation in surrounding tissues Numerous adult flukes 555 (Hapalotrema pambanensis) were recovered from the heart and major vessels HE stain, 556 scale bar = 1.7 mm M AN U SC RI PT 544 AC C EP TE D 557 19 ACCEPTED MANUSCRIPT 2, 31 12, 13, 4, 23, 23 1, 0, 34 13, 14, 5, 22, 28 0, 1, 1, 2, 0, 7, 0, 0, 2, 3, 0, 7, 1, 0, 0, 51 16, 19, 5, 36, 43 1, 1, 1, Small immature Large immature Mature Total 0, 6, 12, 12, 0, 21, 9, 21, 19, 23, 22, 18, 1, 14, 22, 8, 1, 21, 8, 20, 20, 22, 23, 19, 1, 15, 20, 10, 0, 7, 1,2 7, 6, 6, 6, 5, 0, 1, 7, 5, 7, 2, 5, 7, 6, 7, 5, 0, 4, 7, 5, 35, 16 11, 32, 12 33, 14 34, 11 36, 14 29, 1, 20, 34, 12 20, TE D Age group M AN U Poor - Very Poor 11, 14, 10 11, 14, 10 11, Thyroid 14, 10 Spleen 1, Salt gland Gonads 1, Parathyroid GIT 0, Pancreas Gall bladder 13, 20 Lung Fibropapilloma 1, Liver Cornea 6, Kidney Brain 4, RI PT Bladder Good - Fair Heart Aorta 20 Body Condition SC Adrenal gland Table Tissue samples collected from turtles, summarised by age group and body condition Total turtles 558 Total turtles refers to the total number of turtles in each category that underwent gross post mortem examination The first number in each case denotes the 560 number of samples examined histologically, while the second number indicates samples tested by T-RFLP to detect and characterise spirorchiid species 561 Samples that failed to amplify either spirorchiid or Eukaryote 18S DNA have been omitted Abbreviations: GIT = gastrointestinal tract AC C EP 559 ACCEPTED MANUSCRIPT 2 1 N Y 1 EP TE D 84 5 RI PT M AN U N Pancreas Lung Liver Heart 23* Notes H postorchis H pambanensis (A, H) H postorchis (A), H pambanensis (BCx1, Hx1), Carettacola sp (BCx1), C hawaiiensis (P) H postorchis (A) H postorchis (A) Carettacola sp (BC, Li), Neospirorchis Gen (H) C hawaiiensis H pambanensis, Novel species H pambanensis (H) Neospirorchis Gen C hawaiiensis H pambanensis x1, L learedi x2, Neospirorchis Gen x1 H pambanensis Carettacola sp Neospirorchis Gen H postorchis (Ax2), H pambanensis (Ax4, Hx4), L learedi (Hx1, Lu), Carettacola sp (BCx1), Neospirorchis Gen (BCx1, Bx5) SC 10 23* AC C Small immature, very poor Small immature, very poor Small immature, poor Small immature, fair Small immature, very poor Adult, fair Small immature, poor Small immature, very poor Small immature, very poor Small immature, poor Large immature, poor Adult, poor Small immature, poor Small immature, good Body cavity Host characteristics (age, body condition) Small immature, poor Adult, poor Brain Table Summary of adult flukes collected Aorta 562 563 All totals are estimates based on counts of intact worms and fragments * indicates combined total between two organs Column headed ‘notes’ provides 564 details of species present in each organ and an estimate of the total number of adult flukes recovered Abbreviations: N = numerous M = multiple A = aorta, 565 B = brain, BC = body cavity, H = heart, Li = liver, Lu = lung 566 21 ACCEPTED MANUSCRIPT Heart Kidney Liver Lung Pancreas Spleen Thyroid 12 50 29 55 57 75 75 71 13 0 18 0 17 50 88 100 100 100 82 93 100 100 100 13 12 14 11 13 12 50 46 25 21 36 15 50 50 33 43 2 16 100 37 100 100 56 0 0 100 100 93 100 2 39 Hapalotrema postorchis 50 13 Hapalotrema pambanensis 50 Hapalotrema synorchis 0 Learedius learedi 0 Hapalotrema mistroides 0 Amphiorchis sp 0 Hapalotrema Carettacola Neospirorchis Total T-RFLP tested - Hapalotrema T-RFLP Positive (%) Carettacola hawaiiensis Carettacola Gen Total T-RFLP tested - Neospirorchis 21 50 50 31 33 14 27 31 50 58 33 0 0 0 0 0 0 15 50 50 17 23 25 17 0 50 0 15 25 33 0 0 0 0 0 0 EP AC C Total T-RFLP tested - Carettacola TE D T-RFLP Positive (%) M AN U PCR positive (%) SC Total samples RI PT 14 GIT 11 GB 14 FP 12 Brain Bladder Gonads Table Occurrence of spirorchiids in each organ, by genera and by species Aorta 567 2 41 2 16 12 14 11 14 11 0 0 13 0 18 0 0 0 0 0 0 0 0 2 43 2 16 10 14 10 14 11 22 ACCEPTED MANUSCRIPT T-RFLP Positive (%) 50 30 50 19 33 50 43 20 21 50 27 43 Neospirorchis Gen 100 100 95 100 50 88 100 100 100 80 93 100 100 100 Neospirorchis Gen 0 0 0 21 50 Average no Hapalotrema spp present 0 1 1 Average no Carettacola spp present 0 0 0 0 0 0 0 Average no Neospirorchis spp present 1 1 2 2 1 2.5 1.8 2.5 2.5 2.2 1.3 2.1 2.1 2.5 2.8 3.1 2.5 SC M AN U Average total species present RI PT Neospirorchis Gen Total samples refers to total samples available for molecular testing Total PCR positive refers to number of samples that were positive for each genus/group 569 of genera based on first round multiplex PCR results 'Total T-RFLP results' columns refer to total number of samples that were successfully characterised by 570 T-RFLP, or had a validated negative result on multiplex PCR Abbreviations: GIT - Gastrointestinal tract; GB = Gall bladder; FP = Fibropapilloma AC C EP TE D 568 23 ACCEPTED MANUSCRIPT Bladder Brain Cornea Fibropapilloma Gall bladder GIT Gonads Heart Kidney Liver Lung Pancreas Parathyroid Salt gland Spleen Thyroid Total Average no species None observed 7 15 0 13 17 17 12 14 135 1.6 (42) - Mild 10 10 11 82 2.6 (22) - Mild/moderate 0 0 1 3 37 3.5 (8) - Moderate 0 1 33 3.5 (3) – Moderate/severe 0 0 - Severe 10 0 0 Unclassified 0 0 0 SC 0 0 0 0 0 2.0 (1) 0 0 0 14 3.5 (4) 0 0 0 0 0 na (0) Total examined 16 19 36 1 26 26 28 28 30 22 14 26 15 304 Gross lesions are not included in these figures Numbers in brackets denote number of samples with full molecular characterisation of spirorchiid assemblages na TE D 574 EP 573 AC C 572 M AN U Granuloma severity RI PT Aorta Table Summary table of occurrence and severity of trematode ova associated granulomas in tissues examined by histology Adrenal gland 571 24 ACCEPTED MANUSCRIPT Table Results of generalised linear models analysing the effects of variables on granuloma formation in the brain Age OR 2.17 CI 0.45 - 10.44 SE 1.74 P 0.34 OR 0.62 CI 0.07 - 5.16 SE 0.67 P 0.66 Sex 0.96 0.21 - 4.34 0.74 0.96 0.52 0.07 - 4.06 0.54 0.53 Body condition 1.04 0.23 - 4.70 0.80 0.96 1.96 0.22 - 17.34 2.12 0.55 Multispecies infection 16.20 1.57 - 167.74 19.32 0.02 20.10 1.62 - 250.14 25.86 0.02 M AN U 576 Multivariate analysis RI PT Univariate analysis SC 575 Abbreviations: OR = Odds ratio, CI = 95% confidence interval, SE = standard error, P = P-value (0.05 significance limit) AC C EP TE D 577 25 AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Highlights First species-level molecular study of spirorchiidiasis • Eight genotypes detected across fourteen tissue types • Species investigated in terms of tissue tropisms and pathology • Granulomas and arteritis/thrombosis most common lesions • Number of species present correlated with presence and severity of lesions AC C EP TE D M AN U SC RI PT • ... MANUSCRIPT ACCEPTED MANUSCRIPT Molecular epidemiology and pathology of spirorchiid infection in green sea turtles (Chelonia mydas) Phoebe A Chapmana, Helen Owena, Mark Flinta,b, Ricardo J Soares Magalhãesc,d,... Flint M., Eden P.A., Limpus C.J., Owen H., Gaus C., Mills P., 2015b Clinical and pathological findings in green turtles (Chelonia mydas) from Gladstone, Queensland: Investigations of a stranding... overall infection rate of 98% on the basis of histopathology Spirorchiids were also found to be the 66 most common cause of death (41.8%) in Queensland green sea turtles between 2006 and 2009 (Flint